A novel experimental model for the study and evaluation of experimental vaccines to Actinobacillus pleuropneumoniae

Veterinary Immunology and Immunopathology, 41 (1994) 375-380 0165-2427/94/$07.00 © 1994 - Elsevier Science B.V. All rights reserved

375

Short Communication A novel experimental model for the study and evaluation of experimental vaccines to

Actinobacillus pleuropneumoniae J.A. Montaraz*, M.E. Rosales, E. Bautista, G. Barcenas, V. Lara Facultad de Estudios Superiores-Cuautitldn (UNAM), Coordinacirn Generalde Estudios de Posgrado, Apartado PostalNo. 25, Cuautitldn Izcalli, Edo. de Mexico 54700, Mexico

(Accepted 8 September 1993)

Abstract A novel experimental model to study immune protection to Actinobacillus pleuropneumoniaein the rat is described. One-week-old rats born from immunized mothers were challenged intraperitoneally with a suspension ofA. pleuropneumoniaeserotype 1 and mortality recorded up to 48 h postinfection. Immunization with inactivated whole cells ( IWC ) or a whole cell extract (WCE) from A. pleuropneumoniae serotype 1 resulted in protection against an homologous challenge, particularly in the case of WCE where protection was observed at the highest challenge dose of approximately 1000 50% lethal doses.

Abbreviations BHI, brain-heart infusion; cfu, colony forming units; i.p., intraperitoneal; IWC, inactivated whole cells; LD, lethal dose; NAD, adenosine dinucleotide; OD, optical density; PCP, porcine contagious pleuropneumonia; WCE, whole cell extract.

Introduction Actinobacillus p l e u r o p n e u m o n i a e is the etiological agent o f p o r c i n e c o n t a gious p l e u r o p n e u m o n i a ( P C P ) . T w e l v e serotypes o f this b a c t e r i u m h a v e b e e n d e s c r i b e d ( N i e l s e n , 1986 ). T h i s a n t i g e n i c d i v e r s i t y seems e v e n m o r e critical w h e n it is c o n s i d e r e d t h a t pigs r e c o v e r e d f r o m n a t u r a l i n f e c t i o n d e v e l o p an h e t e r o l o g o u s i m m u n i t y while pigs i m m u n i z e d with b a c t e r i n s b e c o m e serot y p e - s p e c i f i c i m m u n e ( R o s e n d a l et al., 1988 ). It seems t h a t toxins, particularly h e m o l y s i n , m a y a c c o u n t for a g o o d p a r t o f the p o l y v a l e n t i m m u n i t y seen a f t e r n a t u r a l i n f e c t i o n , n o t o n l y b e c a u s e this f a c t o r is i n v o l v e d in p a t h o g e n -

*Corresponding author. SSDI0165-2427(93)05250-I

376

z n . Montaraz et aL / Veterinary lmmunology and lmmunopathology 41 (1994) 375-380

esis but also because only two varieties have been associated with all serotypes (Kamp et al., 1991 ). However, it seems feasible to envisage structural components shared by different serotypes and to presume that some may be involved in a polyvalent immune response. To look into this aspect experimentally, the present study proposes an animal model based on the intraperitoneal (i.p.) challenge of 1-week-old rats born from immunized mothers. Although PCP is a disease restricted to lungs and pleura, studies concerned with immunization of pigs make use of systemic routes of immunization, such as subcutaneous or intramuscular, eliciting the most likely antibodies of the IgG and IgM class, which would eventually reach the lungs and exert their protective activity. The model proposed here focuses on the effect of circulating antibodies (IgG and IgM) transferred from mother to young on systemic challenge. The argument here is that if antibodies to certain specificity are protective in the rat, they could also be protective in the pig. It is not known if an i.p. inoculation ofA. pleuropneumoniae in 1-week-old rats results in lesions in the lung. However, other workers using rodents, such as mice and guinea pigs, which were inoculated i.p. observed either lung lesions (Kume and Nakai, 1988 ) or the presence ofA. pleuropneumoniae antigen in the lungs (Komal and Mittal, 1990 ). Material and methods

Bacteria Actinobacilluspleuropneumoniae serotype 1 reference strain 4074 (ATCC No. 27088 ) was kept frozen in liquid nitrogen as a thick suspension in fetal bovine serum. Cultures were prepared from the frozen stock each time bacteria were needed. Experimental vaccines Two different vaccines were tested: inactivated whole cells (IWC) and a whole cell extract (WCE) from A. pleuropneumoniae serotype 1. To prepare IWC, A. pleuropneumoniae was grown on brain-heart infusion (BHI) agar supplemented with 0.001% adenosine dinucleotide (NAD). Cultures were incubated overnight at 37°C and cells were harvested by gently washing with sterile saline (0.18% NaC1). The suspension was adjusted to an optical density (OD) of 1 at 590 nm wavelength (equivalent to approximately 101° colony forming units (cfu) ml-1). The suspension was centrifuged at 1000 × g for 15 min and the cells resuspended and adjusted to 108 cfu ml-1 in saline containing 0.3% formaldehyde. To prepare WCE, bacteria were grown and harvested as above and the suspension subjected to 5 min sonication, 18 000 cycles s- 1 (Ultrasonic Homog-

J.A. Montaraz et al. / Veterinary Immunology and lmmunopathology 41 (1994) 375-380

377

enizer, Cole Parmer, Chicago, IL, USA). The sonicate was centrifuged at 1000 X g for 15 rnin and the supernatant concentrated by incubation at - 20 ° C for 15 min with five volumes of cold acetone. The extract was further centrifuged at 1000 X g for 15 min, the pellet resuspended in 1 ml of sterile saline and the protein concentration estimated by the Bradford method (Bradford, 1976).

Challenge suspension Actinobacillus pleuropneumoniae serotype 1 was cultured, harvested and adjusted to OD 1 as indicated for IWC. The suspension was diluted with sterile saline to reach the desired dose and rats inoculated intraperitoneally with 0.5 ml each. To confirm the dosage, viable counts were performed after inoculation. Establishment of A. pleuropneumoniae serotype 1 50% lethal dose for 1week-old rats Groups of 1-week-old rats (usually from the same litter) were inoculated i.p. with 0.5 ml of a bacterial suspension containing from 10 to 10 lO cfu per rat. Mortality was recorded up to 48 h postinoculation and the 50% lethal dose (LD 50%) calculated by the m e t h o d of Reed and Munch ( 1976 ).

Experimental model The model consisted of immunizing adult female Wistar rats (250 g body weight) with either 500 #g of WCE or 108 cfu m l - ~ of IWC intramuscularly on Days 1 and 15. The first immunization was emulsified (50% v / v ) in Freund's incomplete adjuvant (Difco, Grand Island, USA); the second immunization did not contain adjuvant. Rats were individually mated on Day 8 and their progeny challenged i.p. at 1 week of age. Mortality was recorded up to 48 h postinfection (moribund animals were killed by inhalation of chloroform). N o n - i m m u n i z e d rats were always kept so their progeny could be used as controls. Fisher's exact test was used to establish statistical significance between control and i m m u n i z e d groups.

Results

Establishment of A. pleuropneumoniae serotype 1 LD 50%for I-week-old rats The percentage of survival for the different bacterial dosages was: 10-106 cfu per rat from 100% to 46%; 107-101° from 25% to 0%. With these data, the LD 50% was calculated to be 10 6"672.

37 8

J.A. Montaraz et aL / Veterinary Immunology and Immunopathology 41 (1994) 375-380

Table 1 Effect of immunization of female adult rats upon challenge of their progeny with Actinobacillus pleuropneumoniae

CFU per rat

Number dead/total (% survival) Non-immunizedcontrol

Immunized

3/3 5/5 5/5 4/5

(0) (0) (0) (20)

0/9 (100)* 5/10 (50)* 3/10 (70)* 3/10 (70)*

5/6 (17) 6/6 (0) 6/6 (0)

4/6 (33) 3/6 (50)** 6/6 (0)

W h o & cell extract

107 10s 109 101°

Inactivated w h o ~ cel~

107 108 109

Differences between controls and immunized were significant at *P< 0.05, **P< 0.01.

Effect of immunization with IWC or WCE The effect of immunization of female rats with either WCE or IWC on challenge of their progeny with A. pleuropneumoniae serotype 1 is presented in Table 1. It is observed that immunization with WCE is highly effective even at the higher challenge dose of 101° cfu per rat. With respect to IWC, protection was observed up to the challenge dose of 108 cfu per rat. Discussion

Actinobacilluspleuropneumoniae infection continues to be a problem in pig farming, and among the aspects which require a solution is prevention through vaccination. Much work has been done recently with exotoxins, particularly hemolysin, and this factor seems to be a good candidate for a polyvalent vaccine ( K a m p et al., 1992 ). Nevertheless, since serotype-specificity resides on capsular antigens (Nielsen, 1986), it seems plausible to presume the existence of structural components c o m m o n to more than one serotype. To look into the possible immunogenic value of such components, the present study proposes the challenge of baby rats born from i m m u n i z e d mothers as an experimental model. As a first attempt, IWC and WCE were tested against homologous challenge. Significant levels of protection were observed in the case of the WCE. Preliminary studies, in which rats were i m m u n i z e d with a WCE from A. pleuropneumoniae serotype 7 and their progeny challenged with serotype 1 (data not shown), indicated that the model may also be useful for cross-protection studies.

J.A. Montaraz et al. / VeterinaryImmunology and Immunopathology 41 (1994) 375-380

379

Others have used experimental models to study both the infection and immune protection to A. pleuropneumoniae. Most investigations use mice as the experimental animal (Sebunya and Saunders, 1982; Komal and Mittal 1990; Bhatia et al., 1991 ) but other laboratory species such as guinea pigs have been employed (Kume and Nakai, 1988 ). The idea of using the rat originated from studies concerned with Haemophilus influenzae (A. pleuropneumoniae used to belong to the genus Haemophilus) in which 8-day-old rats were challenged after, for example, passive immunization with monoclonal antibodies to capsular antigens (Hansen et al., 1982). In the case of A. pleuropneumoniae discussed here, an adaptation of the above mentioned model was tried, so that natural passive immunity rather than artificial passive immunity was evaluated. It was interesting that protection conferred by a bacterial extract was better than that elicited by a whole cell bacterin. The obvious explanation seems to be that the response to surface exposed antigens is not as strong as that induced by a mixture of internal and external antigens. Also it is important to consider that WCE is constituted mostly by proteins which, as a whole, display higher antigenicity than polysaccharides which presumably formed most of the exposed antigens in an A. pleuropneumoniae bacterin. In any case, this type of result is encouraging since the final aim of this kind of study is to identify structural components (preferentially proteins) which, perhaps when artificially linked to an exotoxin, such as hemolysin, may constitute a strong immune stimulus resulting in polyvalent protection.

Acknowledgement We thank Jane Galina for reviewing the manuscript.

References Bhatia, B., Mittal, K.R. and Frey, J., 1991. Factors involved in immunity against Actinobacillus pleuropneumoniae in mice. Vet. Microbiol., 29:147-158. Bradford, M.N., 1976. A rapid and sensitive method for the quantitation of microquantities of protein. Anal. Biochem., 72: 248-254. Hansen, E.J., Gulig, P.A., Robertson, S.M., Prish, C.F. and Haanes, E.J., 1982. Immunoprotection of rats against H a e m o p h i l u s influenzae type B disease mediated by monoclonal antibody a H a e m o p h i l u s - o u t e r - m e m b r a n e protein. Lancet, 13: 366-376. Kamp, E.M., Johan, K.P., Anakotta, J. and Smits, M.A., 1991. Identification of hemolytic and cytotoxic proteins ofActinobacillus pleuropneumoniae by use of monoclonal antibodies. Infect. Immun., 59 (9): 3079-3085. Kamp, E.M, Vermeulen, T.M.M., Cruysen, T., van Dijk, J.E., Riepema, K., Smits, M.A. and van Leeuwen, J.M., 1992. Actinobacillus pleuropneumoniae cytolysins: Cross protective effect of a crude cytolysin-vaccine. In! Proceedings of the International Pig Veterinary Society, 1-20 August 1992, The Hague, Netherlands. International Pig Veterinary Society, Netherlands, p. 209.

380

J.A. Montaraz et al. / Veterinary Immunology and lrnmunopathology 41 (1994) 375-380

Komal, J.P.S. and Mittal, K.R., 1990. Grouping ofActinobacillus pleuropneumoniae strains of serotypes 1 through 12 on the basis of their virulence in mice. Vet. Microbiol., 25: 229-240. Kume, K. and Nakai, T., 1988. Efficacy of a bivalent vaccine containing serovar 2 and 5 strains of Haemophilus pleuropneumoniae in pigs or in guinea pigs. Jpn. J. Vet. Sci., 50 ( 1 ): 237241. Nielsen, R., 1986. Serological characterization ofActinobacillus pleuropneumoniae strains and proposal of a new serotype: serotype 12. Acta Vet. Scand., 26: 453-455. Reed, L.J. and Munch, H., 1976. A simple method of estimating 50 percent endpoints. Am. J. Hyg., 27: 493-497. Rosendal, S., Carpenter, W.R. and Wilson, M.R., 1988. Vaccination against pleuroneumonia in pigs caused by Haemophilus pleuropneumoniae. Can. Vet. J., 22:34-35. Sebunya, T.N.K. and Saunders, J.R., 1992. Studies on immunity to Haemophilus pleuropneurnoniae infections in mice. Am. J. Vet. Res., 43 (10): 1793-1798.